Accurately measuring small objects or other physical phenomena is a goal that is pursued in many diverse fields of scientific endeavor. For example, in the study of cellular biology and cellular structures, examining the structural features of cells is essential for many clinical and laboratory studies. The most common tool used in the examination for the study of cells has been the microscope. Although microscope examination has led to great advances in understanding cells and their structure, it is inherently limited by the artifacts of preparation. The characteristics of the cells can only been seen at one moment in time with their structure features altered because of the addition of chemicals. Further, invasion is necessary to obtain the cell sample for examination.
Thus, light scattering spectrography (LSS) was developed to allow for in vivo examination applications, including cells. The LSS technique examines variations in the elastic scattering properties of cell organelles to infer their sizes and other dimensional information. In order to measure cellular features in tissues and other cellular structures, it is necessary to distinguish the singly scattered light from diffuse light, which has been multiply scattered and no longer carries easily accessible information about the scattering objects. This distinction or differentiation can be accomplished in several ways, such as the application of a polarization grating, by restricting or limiting studies and analysis to weakly scattering samples, or by using modeling to remove the diffuse component(s).
LSS has received much attention recently as a means for probing cellular morphology and the diagnosing of dysplasia. The disclosures of the following references are incorporated by reference in their entirety: Backman, V., V. Gopal, M. Kalashnikov, K. Badizadegan, R. Gurjar, A. Wax, I. Georgakoudi, M. Mueller, C. W. Boone, R. R. Dasari, and M. S. Feld, IEEE J. Sel. Top. Quantum Electron., 7(6): p. 887 893 (2001); Mourant, J. R., M. Canpolat, C. Brocker, O. Esponda-Ramos, T. M. Johnson, A. Matanock, K. Stetter, and J. P. Freyer, J. Biomed. Opt., 5(2): p. 131 137 (2000); Wax, A., C. Yang, V. Backman, K. Badizadegan, C. W. Boone, R. R. Dasari, and M. S. Feld, Biophysical Journal, 82: p. 2256 2264 (2002); Georgakoudi, I., E. E. Sheets, M. G. Muller, V. Backman, C. P. Crum, K. Badizadegan, R. R. Dasari, and M. S. Feld, Am J Obstet Gynecol, 186: p. 374 382 (2002); Backman, V., M. B. Wallace, L. T. Perelman, J. T. Arendt, R. Gurjar, M. G. Muller, Q. Zhang, G. Zonios, E. Kline, T. McGillican, S. Shapshay, T. Valdez, K. Badizadegan, J. M. Crawford, M. Fitzmaurice, S. Kabani, H. S. Levin, M. Seiler, R. R. Dasari, I. Itzkan, J. Van Dam, and M. S. Feld, Nature, 406(6791): p. 35 36 (2000); Wax, A., C. Yang, M. Mueller, R. Nines, C. W. Boone, V. E. Steele, G. D. Stoner, R. R. Dasari, and M. S. Feld, Cancer Res, (accepted for publication).
As an alternative approach for selectively detecting singly scattered light from sub-surface sites, low-coherence interferometry (LCI) has also been explored as a method of LSS. LCI utilizes a light source with low temporal coherence, such as broadband white light source for example. Interference is only achieved when the path length delays of the interferometer are matched with the coherence time of the light source. The axial resolution of the system is determined by the coherent length of the light source and is typically in the micrometer range suitable for the examination of tissue samples. Experimental results have shown that using a broadband light source and its second harmonic allows the recovery of information about elastic scattering using LCI. LCI has used time depth scans by moving the sample with respect to a reference arm directing the light source onto the sample to receive scattering information from a particular point on the sample. Thus, scan times were on the order of 5-30 minutes in order to completely scan the sample.
More recently, angle-resolved LCI (a LCI) has demonstrated the capability of obtaining structural information by examining the angular distribution of scattered light from the sample or object under examination. The a/LCI technique has been successfully applied to measuring cellular morphology and to diagnosing intraepithelial neoplasia in an animal model of carcinogenesis, a/LCI is another means to obtain sub-surface structural information regarding the size of a cell. Light is split into a reference and sample beam, wherein the sample beam is projected onto the sample at different angles to examine the angular distribution of scattered light. The a/LCI technique combines the ability of (LCI) to detect singly scattered light from sub-surface sites with the capability of light scattering methods to obtain structural information with sub-wavelength precision and accuracy to construct depth-resolved tomographic images. Structural information is determined by examining the angular distribution of the back-scattered light using a single broadband light source is mixed with a reference field with an angle of propagation. The size distribution of the cell is determined by comparing the osciallary part of the measured angular distributions to predictions of Mie theory. Such a system is described in Cellular Organization and Substructure Measured Using Angle-Resolved Low-Coherence Interferometry, Biophysical Journal, 82, April 2002, 2256-2265, incorporated herein by reference in its entirety.
The a/LCI technique has been successfully applied to measuring cellular morphology and to diagnosing intraepithelial neoplasia in an animal model of carcinogenesis, Such a system is described in Determining nuclear morphology using an improved angle-resolved low coherence interferometry system in Optics Express, 2003, 11(25): p. 3473-3484, incorporated herein by reference in its entirety. The a/LCI method of obtaining structural information about a sample his been successfully applied to measuring cellular morphology in tissues and in vitro as well as diagnosing intraepithelial neoplasia and assessing the efficacy of chemopreventive agents in an animal model of carcinogenesis. a/LCI has been used to prospectively grade tissue samples without tissue processing, demonstrating the potential of the technique as a biomedical diagnostic.
Another technique is optical coherence tomography (OCT). OCT has been established as an excellent technique for cross-sectional imaging of biological samples with high resolution, speed, and sensitivity W. In recent years, several specialized extensions of OCT have been developed in order to gain functional information about probed samples [2-5]. One such extension, which seeks to analyze depth-resolved spectroscopic information about experimental samples, is known as spectroscopic OCT (SOCT) when applied as an imaging technique [2, 6] and Fourier domain low coherence interferometry (fLCI) when applied as an analysis method [7, 8]. Because the spectral scattering and absorption properties of an experimental sample vary depending on its molecular makeup, SOCT obtains increased contrast and functional information by spatially mapping spectral characteristics onto coherence gated images.
In order to generate depth resolved spectroscopic information from data collected in a single domain, SOCT typically employs a short time Fourier transform (STET) or a continuous wavelet transform (CWT), The resulting depth-wavelength distributions are analogous to time-frequency distributions (TEDs) which have been analyzed extensively in the signal processing literature [9, 10], but only recently analyzed in the context of SOCT [11, 12]. Graf and Wax used the Wigner TED from Cohen's class of functions [13] to show that temporal coherence information contained in the Wigner TED cross-terms can be utilized to gain structural knowledge of samples via SOCT signals [12]. However, TEDs generated by the STET are severely limited by the relationship between time and frequency which results in an inherent tradeoff between time (depth) resolution and frequency (wavelength) resolution.
Work in the fields of signal processing and quantum physics have paved the way for a new SOCT processing technique that ameliorates the detrimental effects of the time-frequency resolution tradeoff. Thomson, for example, developed a method particularly well suited for stationary Gaussian signals using orthogonal windows as means for estimating weighted averages for spectral approximations to achieve high-resolution spectral information [9]. Later, Bayram and Baraniuk expanded on Thomson's method by implementing two Hermite-function-based windows to provide a robust analysis of the time-varying spectrum of non-stationary signals, Which are pertinent to fields such as radar, sonar, acoustics, biology, and geophysics [10]. More recently, Lee et al [14] showed that using multiple windows simultaneously can avoid a similar resolution tradeoff in measurement of the position and momentum of a light field.
In burn injuries, treatment of burn wounds depends on the depth of the injury. Superficial 2nd degree burns leave viable keratinocytes in dermal structures such as hair follicles and sweat glands which can regenerate skin. However, deep 2nd degree burns that penetrate to the deep dermis destroys hair follicles and sweat glands and thus, require surgical excision. Determining which skin should be excised and which should be preserved is a key goal in burn care that improves outcomes and reduces costs. In the United States, there are over 2 million burns reported each year and in the military 5-20% of injuries are thermally related. Burn injuries are estimated to cost approximately 7.5 billion/year. Currently, clinical judgment of burn depth is only 70% accurate. Currently, there are no imaging techniques that can penetrate the full skin on the millimeter scale to provide specific diagnostic information that is clinically useful.
In deep spectroscopic imaging of biological samples, tissue scattering limits the penetration depth of most optical imaging techniques by both attenuating the ballistic signal and obscuring it beneath a diffuse background signal. Optical imaging techniques which image with ballistic photons must have a way to separate the desired signal from the diffuse background signal, to image features in a scattering medium, such as tissue.
The optical imaging techniques such as confocal microscopy and optical coherence tomography (OCT) seek to reject multiple scattered light in favor of detecting ballistic light. Confocal microscopy uses a pinhole to accept only light from a given focal plane and reject out of focus light, and is effective to about 3 scattering mean free paths, or approximately 200 μm in tissue. Optical coherence tomography (OCT) and other low coherence interferometry methods (LCI) use interferometry to gate backscattered photons by optical path length, thus isolating photons arriving from a specific depth. OCT is sensitive to about 27 scattering mean free paths or about 1-2 millimeters in tissue. Multiply scattered Low Coherence Interferometry (MS/LCI) enables imaging up to 90 scattering mean free paths or approximately 1 cm in tissue, with 1 millimeter resolution.
However, it uses a time domain detection scheme and requires extensive averaging to achieve this benchmark, leading to data acquisitions in the range of 10-100 minutes and no demonstration of applicability to biological tissues. Further, spectral domain detection suffer from an inherent tradeoff between imaging range (depth) and spectral bandwidth.